Alzheimer's Disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in ethnic groups worldwide and presents a major present and future public health problem.
The brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles of paired helical filaments, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD.
Neurofibrillary tangles are primarily composed of aggregates of hyper-phosphorylated tau protein. The main physiological function of tau is microtubule polymerization and stabilization. The binding of tau to microtubules takes place by ionic interactions between positive charges in the microtubule binding region of tau and negative charges on the microtubule lattice (Butner and Kirschner, J Cell Biol. 115(3):717-30, 1991). Tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with the primary function of tau. Tau that is bound to the axonal microtubule lattice is in a hypo-phosphorylation state, while aggregated tau in AD is hyper-phosphorylated, providing unique epitopes that are distinct from the physiologically active pool of tau (Iqbal et al., Curr Alzheimer Res. 7(8): 656-664, 2010).
The progression of tauopathy in an AD brain follows distinct spreading patterns. A tauopathy transmission and spreading hypothesis has been described based on the Braak stages of tauopathy progression in the human brain and tauopathy spreading after tau aggregate injections in preclinical tau models (Frost et al., J Biol Chem. 284:12845-52, 2009; Clavaguera et al., Nat Cell Biol. 11:909-13, 2009). It is believed that tauopathy can spread in a prion-like fashion from one brain region to the next. This spreading process would involve an externalization of tau seeds that can be taken up by nearby neurons and induce further tauopathy.
Fragments of tau protein in the neurofibrillary tangles move to the cerebrospinal fluid (CSF) where they can be harvested by lumbar puncture and measured by sensitive assays. The presence of neurological disease can thus be detected using assays that recognize tau protein-derived fragments in CSF. Such tau assays require the ability to recognize tau species characteristic of a neurodegenerative condition. Multiply-phosphorylated tau is the leading example of AD-associated tau protein. Therefore, assays that detect multiply-phosphorylated tau protein in CSF may be most effective in detecting the presence of AD.
Phosphorylation is not the only posttranslational modification to consider in measuring tau. Recent studies have demonstrated that in CSF, tau protein exists primarily as fragments rather than as full-length protein (Meredith et al. PLoS One. 8(10):e76523, 2013). Further, the tau fragmentation pattern may be influenced by disease, as proteolysis is frequently aberrant in pathological conditions. Consequently, tau-based assays for neurodegeneration need to provide information not only on the phosphorylation status (e.g. phosphorylation site), but also on the nature of the tau fragments (e.g. length of tau fragment, polarity) that are being measured. However, translation of this idea is hampered by the low endogenous levels of phosphorylated tau, especially in samples from healthy subjects.
In summary, there remains a need for sensitive, precise and accurate methods for detecting multiply-phosphorylated tau in biological fluids. Such methods would be useful to effectively detect, diagnose, stage and track disease progression of neurodegenerative diseases, such as AD and other tauopathies. The methods would also be useful as pharmacodynamics markers for measuring levels of total, free, and therapeutic antibody-bound multiply-phosphorylated tau. The ability to detect and measure multiply-phosphorylated tau fragments is of further importance to the field, as the transmissible tau species may be one or more tau fragments.